FEMS Microbiology Letters 42 (1987) 201-204
Published by Elsevier
201
FEM 02806
A procedure for the selective enrichment
of Halobacteroides halobius and related bacteria
from anaerobic hypersaline sediments
A h a r o n Oren
Division of Microbial and Molecular Ecology, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received 27 January 1987
Accepted 17 February 1987
Key words: Halobacteroides halobius; Enrichment
1. SUMMARY
2. INTRODUCTION
In enrichment cultures for halophilic anaerobic
chemo-organotrophic bacteria from hypersaline
sediments a variety of bacterial types developed.
However, when the sediment samples were first
heated to 80-100°C for 10-20 min, bacteria resembling Halobacteroides halobius were selectively
enriched. Terminal endospores were observed in
some of the cultures of this type of bacterium,
previously unknown to produce endospores.
Halobacteroides-type cells were found not to be
restricted to Dead Sea sediments, but were isolated also from seawater evaporation ponds. The
finding of endospore formation in Halobacteroides
supports its proposed phylogenetic relationship
with other endospore-forming anaerobic halophilic
bacteria.
During the past few years a number of obligately anaerobic, moderately halophilic, fermentative bacteria from hypersaline sediments have
been isolated and characterized [1,2]. Four isolates
were described (Halobacteroides halobius [3],
Haloanaerobium praevalens [4], Clostridium lortetii
[5], and Sporohalobacter marismortui DY-1 (Oren,
A., Pohla, H. and Stackebrandt, E., Syst. Appl.
Microbiol., in press), all requiting salt concentrations between 1 and 4 M [1,2]. The two last-named
strains were shown to have the potency of producing endospores. A comparative study of 16S ribosomal RNA oligonucleotide sequences showed that
the four strains are related to each other, while
being unrelated to any of the major subgroups of
the eubacterial kingdom to which they belong, and
a new family was created to contain the halophilic
anaerobic chemo-organotrophs: the Haloanaerobiaceae [6].
The observation that at least two known
anaerobic halophilic bacteria are able to produce
endospores led us to attempt to selectively isolate
such forms from anaerobic hypersaline environments by means of a negative selection procedure
Correspondence to: A. Oren, Division of Microbial and Molecular Ecology, Institute of Life Sciences, Hebrew University of
Jerusalem, Jerusalem 91904, Israel.
0378-1097/87/$03.50 © 1987 Federation of European Microbiological Societies
202
based on the heat resistance of the endospores. All
experiments thus performed gave rise to development of long, slender rods of the type that was
described earlier as Halobacteroides halobius [3],
an organism in which the formation of endospores
was not previously observed.
The present work describes the selective enrichment and isolation of Halobacteroides-types of
obligately anaerobic halophilic bacteria, based on
the heat resistance of their resting stages.
3. MATERIALS A N D M E T H O D S
3.1. Sediment samples
Hypersaline anaerobic sediments used in this
study were sampled in part from the surroundings
of a saline sulfur spring on the westem shore of
the Dead Sea near Ein Gedi. The water of the
spring has a salinity of around 180 g/1 total
dissolved salts, and a temperature of 39 ° C at the
source. Details on the biota of the spring and on
the chemical properties of the water can be found
elsewhere [2].
Additional sediment samples were taken from
the bottom of evaporation ponds of a commercial
solar salt production facility near Elat, containing
water of varying salinities as specified in the experiments.
3.2. Enrichment experiments
Sediment samples were suspended in a small
quantity of the overlaying water, and portions
(approximately 20-40 mg dry weight) of the resulting slurry were injected into 25-ml culture
tubes provided with a thick rubber stopper [7],
and containing 5 or 10 ml growth medium of the
following composition [3] (g/l): NaC1, 88 or 140;
MgC12 • 6H20, 20.3; CaC12 • 2H20, 7.35; KC1, 3.7;
glucose, 5.0; yeast extract, 5.0; resazurin, 0.001;
L-cysteine-HC1, 0.5, and piperazine-N, N'-bis (2ethanesulfonic acid) (PIPES) to a final concentration of 25 mM, p H 6.5. The glucose and PIPES
buffer were added from concentrated autoclaved
anaerobic solutions to the autoclaved medium.
Media were prereduced by boiling under nitrogen,
whereafter the cysteine was added, and anaerobic
culture techniques as described by Balch et al. [7]
were used throughout. Immediately after inoculation the tubes were submerged in a water bath at
temperatures between 65 and 1 0 0 ° C for varying
periods as specified in the experiments, whereafter
the tubes were cooled, and incubation was proceeded at 37 o C. For comparison inoculated tubes
that did not receive the heat treatment were included in the experiments.
To test for heat resistance of Halobacteroides
halobius cultures, tubes were inoculated with the
type strain of H. halobius (ATCC 35273) or with a
culture isolated in the course of the experiments,
and after different heat treatments as above tubes
were incubated at 37°C, and growth was monitored after 2-3 days.
3.3. Identification of the developing organisms as
Halobacteroides halobius
Bacteria developing in the enrichment cultures
were identified as H. halobius on the basis of
selected properties of the organism as described
[3], such as morphology of young and senescent
cells, hydrogen production, salt requirement, obligate anaerobic metabolism, and substrate
specificity.
4. RESULTS A N D DISCUSSION
When anaerobic hypersaline sediment samples
from the different environments tested (a saline
sulfur spring and evaporation ponds used for the
production of solar salt) were used as an inocuhim
for enrichment cultures, with anaerobic media
containing 8.8 or 14% NaC1 and with glucose and
yeast extract as carbon and energy sources, a
variety of bacteria developed. Characteristic long
slender rod-shaped cells resembling Halobacteroides halobius [3] were observed in most
cultures, but they were generally outnumbered by
smaller motile rod-shaped bacteria. A similar phenomenon was described earlier for the sulfur
spring, from which facultatively anaerobic bacteria
resembling Vibrio costicola and others were isolated in this way [2]. However, when the incubation of the cultures was preceded by a heat treatment ( 8 0 - 1 0 0 ° C for 5-20 min) most cultures
gave rise to mass development of the long slender
203
Fig. 1. Phase-contract micrographs of Halobacteroides
halobius-like bacteria developingin an enrichment culture using
medium with 14% NaC1, inoculated with sediment from the
sulfur spring near Ein Gedi, and pasteurized at 100 °C for 20
min. (A) Young cells. (B) and (C) endospores developingin an
older culture. The bars represent 10/~m.
Halobacteroides-type rods (Fig. 1A), often in pure
culture, sometimes accompanied by shorter rodshaped bacteria. The long rod-shaped bacteria were
found both in samples from the sulfur spring and
in mud samples from the evaporation ponds at all
salinities tested (5.8-15.3%). A comparison of one
of the isolated strains with the type strain of
Halobacteroides halobius [3] showed similar morphological features, a similar salt requirement, obligate anaerobic metabolism in both, and similarity in the range of substrates fermented and products formed.
The observation that Halobacteroides-like
organisms resist heat treatment was unexpected,
as formation of endospores in this organism was
never reported, though two related organisms
(Clostridium lortetii [5] and strain DY-1 [1,2]) are
known to produce endospores. To test for the heat
resistance of growing cells of the slender rods
isolated from one of the pasteurization experiments, a culture in the exponential growth phase
was heated for 5 rain at 65°C, whereafter serial
10-fold dilutions in growth medium were made,
which were incubated at 37 ° C. No viable cells
were recovered from the heat-treated sample, compared to at least 105 viable cells/ml from a control sample that was not heated. Similar results
were obtained using the type strain of Halobacteroides halobius. In several of our cultures terminally swollen cells with developing endospores
were observed (Figs. 1B and C); in other cultures
the early autolysis of the cells [3] probably prevented the development of spores. The results
obtained suggest that Halobacteroides and related
bacteria may survive in the Dead Sea sediments
(having salinities much higher than those supporting growth [3]) as resistant endospores, rather than
as fragile, easily lysing vegetative cells.
The endospores of the Halobacteroides-like cells
from the sediments of the evaporation ponds and
the sulfur spring showed a heat resistance comparable to that of endospores of the genus Bacillus: heating for 20 min at 100 ° C was not sufficient to destroy their viability. Upon heat treatment for 10 min at 121°C in an autoclave no
viable cells were recovered. No further quantitative studies on the heat resistance of the spores
were performed.
Heat resistance was also observed in a recently
isolated halophilic methanogenic bacterium [8].
This organism, growing opumally at 5 0 - 5 5 ° C
with an upper limit at 57 ° C, was reported to grow
after 20 min incubation of the culture at 100 ° C. It
was not stated whether the vegetative cells them-
204
selves are heat-resistant, or whether endospores
m a y be involved (which would be the first reported case of endospore formation in the
Archaebacterial kingdom, with the possible exception of the not well characterized aerobic sporef o r m i n g halophile described as 'Halosporobacterium paris' [9]).
Two of the four previously described representatives of the Haloanaerobiaceae were reported
to form endospores [1,2]. The present study shows
that isolates very similar or identical to Halobacteroides halobius possess heat-resistant spores, thus
increasing the extent of similarity between the
different representatives of the group. Haloanaerobium praeoalens, an anaerobic bacterium abundant in the sediments of the Great Salt Lake,
U t a h [4], and related to Halobacteroides halobius,
Clostridium lortetii and strain DY-1 as evidenced
by 16S ribosomal R N A nucleotide sequence analysis [1,2,6], was never shown to possess endospores. A search for heat-resistant forms in Great
Salt Lake sediments is thus r e c o m m e n d e d in view
of the phylogenetic relationship of Haloanaerobium
with spore-forming obligately anaerobic halophiles.
ACKNOWLEDGEMENTS
I thank Y. C o h e n for help in obtaining sedim e n t samples from the evaporation ponds.
This work was supported by a grant from
H o u s t o n Lighting & Power C o m p a n y , Houston,
TX, under a university participation p r o g r a m
administered by D y n a t e c h R / D C o m p a n y .
References
[1] Oren, A. (1986) FEMS Microbiol. Rev. 39, 23-29.
[2] Oren, A. (1987) in Biotechnology Applied to Fossil Fuels
(Wise, D.L., Ed.). CRC Press, Boca Raton, in press.
[3] Oren, A., Weisburg, W.G., Kessel, M. and Woese, C.R.
(1984) System. Appl. Microbiol. 5, 58-70.
[4] Zeikus, J.G., Hegge, P.W., Thompson, T.E., Phelps, T.J.
and Langworthy, T.A. (1983) Curr. Microbiol. 9, 225-234.
[5] Oren, A. (1983) Arch. Microbiol. 136, 42-48.
[6] Oren, A., Paster, B.J. and Woese, C.R. (1984) Syst. Appl.
Microbiol. 5, 71-80.
[7] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and
Wolfe, R.S. (1979) Microbiol. Rev. 43, 260-296.
[8] Zhilina, T.N. (1986) System. Appl. Microbiol. 7, 216-222.
[9] Fujii, T. (1980) Bull. Jap. Soc. Sci. Fish. 46, 1545.